Tetro reverse transcriptase kit

Total RNA from cultured cells was isolated with Tri-reagent (Thermo Scientific) and using a phenol chloroform method. Contaminating DNA was digested using DNase I RNase-free (Thermo Scientific) treatment. Reverse transcription was carried out on 100 ng total RNA using Moloney Murine leukemia virus reverse transcriptase (M-MLV RT), provided with the Tetro Reverse Transcriptase kit (Bioline, London, UK). cDNA synthesis were performed following the manufacturers protocol, using a T100 Thermo Cycler (Bio-Rad, Hercules, CA, USA). Quantitative RT-PCR was performed in a total volume of 20 µL using the SensiFast SYBR and Fluorescein Kit (Bioline, London, UK). The PCR reaction profile consisted of initial enzyme activation at 95 °C for 10 s, followed by 40 cycles of denaturation at 95 °C for 15 s, annealing 30 s. with temperature dependent on the primer sequences and extended at 72 °C for 30 s. with a single fluorescence measurement. The series of cycles were followed by a melt curve analysis to ensure reaction specificity. The expression level of each gene was normalized to the housekeeping gene, GAPDH. Subsequently, the relative gene expression (Qn) was calculated in relation to the GAPDH gene.

0.5 µg of total RNA were reversely transcribed using the Tetro Reverse Transcriptase kit (Bioline, Luckenwalde, Germany) according to the following protocol: RNA and RNAse free water were mixed in a total volume of 12 µl containing 0.5 µg RNA. 7 µl of reaction mixture (4 µl fivefold Reaction Buffer, 1 µl dNTP’s (10 mM each), 1 µl Oligo dT18 (10 µM), 1 µl RNase Inhibitor (Bioline, Luckenwalde, Germany). 1 µl Tetro reverse transcriptase were added and the samples were incubated for 1 h at 45 ℃. The reaction was terminated by incubating the sample for 5 min at 85 °C.

Reverse transcription was performed with Tetro Reverse Transcriptase kit (Bioline) according to the manufacturer's instructions. Briefly, 2 μg of total RNA was used as templates for each reaction. qPCR products were prepared using a SensiFAST SYBR No‐ROX kit (Bioline) according to the instructions and performed in the Light Cycler 480 (Roche). Primers are listed in Table EV1.